PROJECT SUMMARY The proposed research focuses on defining the molecular regulators of chimeric antigen receptor (CAR) T- cell activity against cancer. CAR therapy has generated great enthusiasm in light of its efficacy in the treatment of B-cell neoplasms, however significant limitations still prevent its broader success. Approximately of patients with non-Hodgkin lymphoma do not respond, reflecting a failure of cytotoxicity, and many patients with acute lymphoblastic leukemia relapse after treatment due to a failure of CAR T-cell persistence. The studies proposed aim to determine how these essential T-cell functions are regulated in CAR T cells. Preliminary data suggest that death receptors, surface proteins that initiate apoptosis, are essential in CAR T- cell cytotoxicity. Using molecular profiling and gene editing of T-cell and tumor samples from Penn's CAR clinical trials, I will define the mechanistic role of death receptors in CAR-driven cytotoxicity and tumor escape. Persistence is mediated by the formation of immune memory, and data show that CARs activate cellular programs that suppress memory formation. The studies outlined in this proposal will utilize xenograft mouse models of human leukemia to determine how CAR activation of the PI3K signaling pathway impedes formation of memory cells and impairs CAR T-cell persistence. Using clinical trial samples, this work will further aim to identify additional molecular pathways that limit persistence using proteomic and phosphoproteomic analysis. The goal of the proposed five-year training program is the development of my independent research career as a physician-scientist focused on cellular immunotherapies. I completed residency training in Internal Medicine and am in my final year of fellowship training in Hematology/Oncology. I am now expanding my scientific expertise in molecular biology and immunology, as well as clinical expertise in allogeneic hematopoietic cell transplantation. My goal for the near future is to develop the skills necessary to drive a distinctive program of scientific investigation. Specifically, I seek to gain expertise in genome editing, proteomics and computational biology through didactic and practical training in order to augment my scientific skillset and establish independence. My long-term goal is to enhance the efficacy of T-cell immunotherapy and improve outcomes for patients with hematologic cancers. I will be mentored by Dr. Carl June, an international leader in cellular immunotherapy who is well equipped to provide me with the mentorship I need to succeed. To add breadth and depth to my scientific and career guidance, I have assembled a Mentoring Committee composed of exceptional scientists. The University of Pennsylvania had led the field of cellular immunotherapy science and clinical practice, and provides the ideal environment for my training as a cell therapy investigator. The proposed studies have the potential to provide critical insight into CAR biology. The application of cutting-edge techniques to interrogate clinical trial samples and dissect the mechanisms limiting CAR activity will identify features that predict success and directly inform the design of more effective CAR therapies.